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Proc Natl Acad Sci U S A. Apr 15, 1992; 89(8): 3419–3423.
PMCID: PMC48879

Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences.

Abstract

We describe an efficient method for the construction of small-insert genomic libraries enriched for highly polymorphic, simple sequence repeats. With this approach, libraries in which 40-50% of the members contain (CA)n repeats are produced, representing an approximately 50-fold enrichment over conventional small-insert genomic DNA libraries. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of Escherichia coli allowed the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymine. This DNA was used as a template for second-strand DNA synthesis, primed with (CA)n or (TG)n oligonucleotides, at elevated temperatures by a thermostable DNA polymerase. Transformation of this mixture into wild-type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil-containing DNA molecules. In this manner, a library highly enriched for the targeted microsatellite-containing clones was recovered. This approach is widely applicable and can be used to generate marker-selected libraries bearing any simple sequence repeat from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest.

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  • Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet. 1980 May;32(3):314–331. [PMC free article] [PubMed]
  • Litt M, Luty JA. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Hum Genet. 1989 Mar;44(3):397–401. [PMC free article] [PubMed]
  • Miesfeld R, Krystal M, Arnheim N. A member of a new repeated sequence family which is conserved throughout eucaryotic evolution is found between the human delta and beta globin genes. Nucleic Acids Res. 1981 Nov 25;9(22):5931–5947. [PMC free article] [PubMed]
  • Hamada H, Kakunaga T. Potential Z-DNA forming sequences are highly dispersed in the human genome. Nature. 1982 Jul 22;298(5872):396–398. [PubMed]
  • Hamada H, Petrino MG, Kakunaga T. A novel repeated element with Z-DNA-forming potential is widely found in evolutionarily diverse eukaryotic genomes. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6465–6469. [PMC free article] [PubMed]
  • Tautz D, Renz M. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res. 1984 May 25;12(10):4127–4138. [PMC free article] [PubMed]
  • Weber JL. Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms. Genomics. 1990 Aug;7(4):524–530. [PubMed]
  • Olson M, Hood L, Cantor C, Botstein D. A common language for physical mapping of the human genome. Science. 1989 Sep 29;245(4925):1434–1435. [PubMed]
  • Green ED, Mohr RM, Idol JR, Jones M, Buckingham JM, Deaven LL, Moyzis RK, Olson MV. Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: application to the mapping of human chromosome 7 using yeast artificial chromosomes. Genomics. 1991 Nov;11(3):548–564. [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. [PubMed]
  • Lindahl T. DNA repair enzymes. Annu Rev Biochem. 1982;51:61–87. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Lawyer FC, Stoffel S, Saiki RK, Myambo K, Drummond R, Gelfand DH. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J Biol Chem. 1989 Apr 15;264(11):6427–6437. [PubMed]
  • Innis MA, Myambo KB, Gelfand DH, Brow MA. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. [PMC free article] [PubMed]
  • Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7276–7280. [PMC free article] [PubMed]
  • Brenig B, Brem G. Direct cloning of sequence tagged microsatellite sites by DNA affinity chromatography. Nucleic Acids Res. 1991 Oct 11;19(19):5441–5441. [PMC free article] [PubMed]
  • Edwards A, Civitello A, Hammond HA, Caskey CT. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am J Hum Genet. 1991 Oct;49(4):746–756. [PMC free article] [PubMed]

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